DE102012103041A1 - New isolated antisense-oligonucleotide comprising sequence that is hybridized to messenger RNA-splicing-sequence of mutation-bearing exons of pre-messenger RNA of titin-gene and induces skipping of exons, used to treat heart disease - Google Patents

Abstract

Isolated antisense-oligonucleotide (AO) comprising a sequence that is specifically hybridizable to a messenger RNA (mRNA)-splicing-sequence of mutation-bearing exons of pre-mRNA of titin-gene and is able to induce a skipping of the exons is new. ACTIVITY : Cardiant; Cardiovascular-Gen. MECHANISM OF ACTION : Angiotensin-converting enzyme inhibitor; Beta-blocker.

Description

The present invention relates to an isolated antisense oligonucleotide, a pharmaceutical composition, and uses and methods employing the isolated antisense oligonucleotide and the pharmaceutical composition.

Dilated cardiomyopathy (DOM) is a pathological enlargement of the heart muscle, especially of the left ventricle. A systolic pumping failure leads to a progressive loss of ejection performance, which is sometimes associated with a life-threatening cardiac arrhythmia. The prognosis of affected patients is unfavorable. The five-year mortality is about 20%. One of the main causes leading to the development of a DOM are mutations in the so-called titin gene, which codes for a protein of the heart and skeletal musculature. More than fifty such DCM-causing titin mutations have since been identified.

A causal treatment of DCM does not currently exist. Affected patients receive symptomatic heart failure therapy and cardiac arrhythmias using ACE inhibitors and beta-blockers.

Against this background, it is the object of the present invention to provide novel compounds, compositions and methods for the treatment of DCM produced by mutations in the titin gene which are based on a causal therapeutic approach.

This object is achieved by an isolated antisense oligonucleotide (AO) having a sequence that is specifically hybridizable to an mRNA splicing sequence of an exon of the pre-mRNA of the titin gene and that is capable of skipping to induce this exon.

The principle of "exon skipping" has hitherto been described in the prior art only in connection with Duchenne muscular dystrophy (DMD). By way of example, reference is made to the following documents from the prior art: Cirak et al. (2011), exon skipping and dystrophin restoration in patients with Duchenne muscular dystrophy after systemic phosphorodiamidate morpholino oligomer treatment: at open-label, phase 2, dose-escalation study, Lancet, Vol. 378, pages 595-605 . EP 1 191 097 . EP 1 619 249 . WO 2006/000057 . EP 1 857 548 . WO 2010/048586 . US 2012/0041050 and US 2012/0022134 , In the case of DMD, a protein that is important for the stability of the muscle membrane, the dystrophin, is not formed at all or in dysfunctional form, which sooner or later leads to the breakdown of muscle fibers and replacement by fat or connective tissue. Responsible are mutations in the dystrophin gene. By using specific AOs mutation-carrying exons of the dystrophin gene will be "skipped" during transcription at the level of pre-mRNA. The result is a shorter but functionally intact protein.

The titin gene, also referred to as the connectin gene, encodes an approximately 3.6 megadalton-sized elastic protein, which is composed of filaments. It consists of over 30,000 amino acids and contains 320 protein domains. Human titin is the largest known human protein. Titin is part of the sarcomere, the smallest functional unit in striated muscle. The purpose of titin in the sarcomere is to center the myosin heads between the actin filaments and return the contactor after stretching.

Therefore, according to the invention, the titin gene is that of a mammal, preferably the human titin gene or that of a mouse.

In humans, the titin gene is located on chromosome 2, 179.1 to 179.38 Mb. The gene ID is 7273 (Entrez). The NCBI reference sequence is NG_011618.2.

In the mouse, the titin gene is located on chromosome 2, 76.51 to 76.64 Mb. The gene ID is 22138. The NCBI reference sequence is NC_000068.7.

According to the invention, an mRNA splicing sequence is understood as meaning such a nucleotide sequence which is involved in mRNA splicing, in particular splice donor sequences, splice acceptor sequences or exonic splicing enhancer sequences. In addition, splicing branch points and exon recognition sequences as well as spice enhancers come into question as mRNA splicing sequences.

As a pre-mRNA or precursor mRNA is a not or incompletely processed eukaryotic mRNA called.

"Skipping" means that the AO according to the invention prevents hybridization to the mRNA splicing sequence of a mutation-bearing exon on the pre-mRNA that this exon is incorporated into the mature mRNA. The corresponding exon is consequently "dumped", i. H. skipped. This results in a shortened mature mRNA coding for a truncated but functional titin.

Specific hybridization means that the AO of the present invention has a nucleotide sequence complementary to regions of the mRNA splicing sequence of the mutation-bearing exon. Accordingly, the hybridization can also take place under stringent conditions and differs from nonspecific accumulations.

The object underlying the invention is hereby completely solved.

The AO of the invention, when introduced into an organism suffering from a dilative cardiomyopathy caused by a mutation in the titin gene, causes the corresponding mutation-carrying exon not to be present in the mature mRNA. The mature mRNA is then translated into a functional titin protein without the mutation-carrying domain or mutation-bearing domain, respectively. As the inventors were able to show in a DCM mouse model, the administration of the AO according to the invention can restore the sarcomere structure.

According to a further development of the invention, the isolated AO has a sequence which is specifically hybridizable to the mRNA splicing sequence of the exon 326 of the titin gene.

The numbering of the exons of the titin gene is not uniform in the scientific literature. The inventors refer to the numbering, such as in Gerull et al. (2002), Mutations of TTN, encoding the giant muscle filament titin, cause familial dilated cause familial dilated cardiomyopathy, Nat. Genet. 30, pages 201 to 204 , is used. According to the ESEMBL nomenclature, the exon 326 of the human titin gene corresponds to the transcript ENST00000342992 (SEQ ID NO: 14), which is designated there as exon 275, the exon 326 of the murine titin gene corresponds to the transcript ENSMUST00000099981 (SEQ ID NO. 15), which is designated there as exon 309.

With this measure, such an AO is provided with which such an exon can be "dumped", which, according to findings of the inventors in an autosomal dominant DCM, which could be detected, for example, in a large Australian family causes a frameshift mutation. Specifically, it is the truncation mutation c.43628insAT; see. Gerull et al. (2002, loc. Cit.) , Patients affected by this mutation can be effectively helped with the preferred embodiment of the invention.

According to a preferred development, the isolated AO according to the invention has a length of about 5 to 50, preferably of about 10 to 40, more preferably of about 15 to 30, and most preferably of about 19 to about 22 nucleotides.

This measure has the advantage that the isolated AO is sufficiently long in order to bind stably to the mRNA splicing sequence of the mutation-carrying exon and to be able to trigger skipping. The splicing process on the mutation-carrying exon and thus the incorporation into the mature mRNA is thus prevented. The specified length ranges have proved to be particularly advantageous according to findings of the inventors.

According to a preferred development according to the invention, the mRNA splicing sequence is an exonic splicing enhancer (ESE) sequence.

This measure has the advantage that the AO according to the invention is developed in such a way that it binds to the crucial mRNA splicing sequence in the exon of the titin gene, and thus initiates skipping of the exon.

According to a preferred development, the AO according to the invention comprises morpholino oligonucleotides and / or 2'-O-methyl phosphorothioate oligonucleotides.

This measure has the advantage that the lipophilicity of the AO according to the invention is increased. The AO according to the invention is thus better absorbed into the affected cells. Furthermore, the stability of the AO according to the invention is increased by the replacement of the natural nucleotides. The half-life is several hours when using morpholino oligonucleotides, and several weeks when using 2'-O-methyl-phosphothiorate oligonucleotides. The morpholino technology is described for example in Sazani et al. (2011), Chemical and mechanical toxicology evaluation of exon skipping phosphorodiamidate morpholino oligomers in mdx mice, Int. J. Toxicol. 30, pages 322-333 , The 2'-O-methyl phosphorothioate technology is described in Heemskerk et. Al (2010), Preclinical PK and PD Studies on 2'-O-Methyl-phosphorothioate RNA Antisense Oligonucleotides in the mdx Mouse Model. Mol. Ther. 18, pages 1210 to 1217.

According to a preferred embodiment, the isolated AO of the invention has a nucleotide sequence specific to at least about 5, preferably at least about 10, more preferably at least about 15, most preferably at least about 19 continuous nucleotides of the sequence SEQ ID NOs / or 2 can hybridize.

This measure has the advantage that the AO according to the invention is provided with such a sequence which corresponds to the ESE sequence of the exon 326 of the human (SEQ ID No. 1) or the mouse titin gene (SEQ ID No. 2) and thereby is able to induce skipping of exon 326.

According to a preferred development, the AO according to the invention has a nucleotide sequence which is selected from the group consisting of SEQ ID Nos. 3 to 13.

It is advantageous in this case that the AO according to the invention has nucleotide sequences which have been successfully tested by the inventors. Sequences SEQ ID NOs: 3 to 7 hybridize to ESE sequences of mouse titin exon 326. The sequences SEQ ID Nos. 8 to 13 hybridize to an ESE sequence of human titin exon 326, so that the corresponding AO according to the invention are suitable for administration to humans.

According to a preferred development, the isolated AO described above is designed for the treatment of dilated cardiomyopathy (DCM).

By this measure, a compound is provided, with which a causal therapeutic treatment of mutations in titin caused dilative cardiomyopathy is possible.

Another object of the invention is a pharmaceutical composition comprising the isolated AO of the invention and a pharmaceutically acceptable carrier.

Pharmaceutically acceptable carriers are extensively described in the art, for example, in U.S. Pat Kibbe et al. (2000), Handbook of Pharmaceutical Excipients, Third Edition, American Pharmaceutical Association and Pharmaceutical Press ,

The invention further relates to the use of the isolated AO according to the invention for the specific inhibition of an mRNA splicing sequence, preferably an ESE sequence, of a mutation-carrying exon of the pre-mRNA of the titin gene, preferably of the exon 326 of the titin gene to induce a skipping of the exon.

The invention further relates to the use of the isolated AO according to the invention or the pharmaceutical composition according to the invention for the treatment of dilated cardiomyopathy (DCM).

Another object of the present invention relates to a method for inducing a skipping of a mutation-carrying exon of the pre-mRNA of the titin gene, preferably of the exon 326 of the titin gene, the administration of the isolated AOs or the inventive pharmaceutical composition of the invention in a biological cell or a living being, such as a mammal or a human.

A further subject matter of the present invention relates to a method for the treatment of dilated cardiomyopathy (DCM), which comprises the administration of the isolated AO according to the invention or the pharmaceutical composition according to the invention into a DCM-affected animal.

The properties, features and advantages of the isolated AOs according to the invention apply equally to the uses and methods according to the invention.

It is understood that the features mentioned above and those yet to be explained below can be used not only in the particular combination given, but also in other combinations or in isolation, without departing from the scope of the present invention.

The invention will now be explained in more detail with reference to embodiments, from which further features and advantages. The embodiments are merely illustrative and do not limit the scope of the present invention.

The accompanying figures show the following:

1 Development of an antisense oligonucleotide (AO) which leads to the skipping of exon 326;

2 Treatment of homozygous titin mouse embryos with the AO;

3 Exon skipping in cultured mouse mouse embryos;

4 Exon skipping in the heterozygous DCM mouse model;

5 Exon skipping on humans.

embodiments

Example 1 - Exon 326 mutation

For a large Australian family, an autosomal dominant inherited dilated cardiomyopathy (DCM) is described. Coupling and haplotype analysis identified a heterozygous insertion mutation of two base pairs in the exon 326 of the titin gene as the molecular genetic cause of DCM (TTN truncation mutation c.43628insAT; Gerull et al. (2002, loc. Cit.) ).

Example 2 - Mouse Model

A knock-in mouse was generated which integrated the human 2 bp insertion mutation in exon 326 into the titin locus. While the homozygous animals die in utero during embryonic development, the heterozygous mice initially develop normally under rest conditions and show no cardiac phenotype. The heterozygous mice develop a DCM under the influence of cardiac stress. The mouse model is described in Gramlich et al. (2009), Stress-induced dilated cardiomyopathy in a knock-in mouse model mimicking human titin-based disease, Journal of Molecular and Cellular Cardiology 47, pp. 352-358 ,

Example 3 - Development of antisense oligonucleotides

In the 1A the strategy for developing an antisense oligonucleotide is shown which leads to skipping of the mutation-bearing exon 326. The mutation in exon 326 is represented by "MUT". The exonic splicing enhancer sequence is shown as "ESE". The antisense oligonucleotide ("AO") of the present invention attaches to the ESE of exon 326 of the pre-mRNA and thereby prevents exon 326 from being present in the mature mRNA. The exon 326 is "dumped".

Table 1 below shows the antisense oligonucleotides generated by the inventors that successfully skipped exon 326 of the mouse titin gene (SEQ ID NOs: 3-7) and humans (SEQ ID NOs: 8-13 ) could be realized.

Tab. 1: Antisense-Oligonucleotide zum Skipping von Exon 326 des Titin-Gens; H = human, M = Maus

Tab. 1: Antisense oligonucleotides for skipping exon 326 of the titin gene; H = human, M = mouse

In the 1B the technical feasibility of the exon skipping of Titin Exon 326 is shown as an example. Murine cardiomyocytes, so-called HL1 cells, were transfected in 5 different batches (1 to 5) with the AO having the sequence SEQ ID No. 3. 24 hours later, the mRNA was isolated and demonstrated by means of RT-PCR, the successful skipping. "C" stands for control and represents the PCR product from the batch without AO. Same results were obtained for the other AOs listed in Table 1.

Example 4 - Treatment of homozygous titin mouse embryos

Homozygous titin-deficient embryos usually die on gestation day 9 due to severe damage in sarcomere formation and cardiac development.

2 shows by way of example the confocal microscopy of a homozygous mouse embryo treated with the AO with the sequence SEQ ID No. 3. Homozygous mouse murine embryos were removed on gestation day 8.5 and continued in culture. A part of the mouse embryos was incubated with the AO. Twenty-four hours later, the mouse embyons were examined functionally, histologically, immunohistologically and electron microscopically.

The treatment with the AO led to the normalization of the sarcomere structure, which is evident by the transverse striation of the red-colored titin filaments. Untreated homozygous mouse embryos show no ordered sarcomere formation; the hearts do not develop contractile function.

Corresponding results were obtained for the treatment with the AOs having the sequences SEQ ID Nos. 4 to 7.

Example 5 - Exon skipping in cultured mouse embryos

3 shows at the electron microscopic level the restoration of a sarcomere structure in homozygous titin-deficient mouse embryos by treatment with the AOs; The treatment with the AO with the sequence SEQ ID No. 3 is shown as an example. Homozygous mouse embryos ("HOM", "KO" stands for "knock-out") were removed on gestation day 8.5 and cultured for a further day (E8.5 + 1, "E" stands for "embryonic stage").

Embryonic mouse hearts usually start on gestation day 9. The homozygous mouse embryos, however, have no ordered sarcomere structure and therefore suggest only very rudimentary. The treatment with the AO according to the invention leads to the restoration of sarcomere formation, which is accompanied by a contraction of the heart ("contractile function").

The more ordered the actin and myosin filaments can be, the thicker the filament width becomes. Homozygous mouse hearts have little actin / myosin assembly, therefore very thin filaments. Treatment with the AO according to the invention leads practically to a normalization of the filament thickness.

Corresponding results were also achieved for the treatment with the AOs having the sequences SEQ ID Nos. 4 to 7.

The experiments of Examples 4 and 5 show that therapy with the AOs according to the invention on the intact organism is possible and extremely effective.

Example 6 - Exon skipping in the heterozygous DCM mouse model

Heterozygous titin mice (HET) usually develop normally and show no cardiac phenotype. However, various cardiac stress models can provoke a DOM in the mouse model. For example, a 14-day application of angiotensin II leads to pronounced heart failure in heterozygous titin-deficient mice; see. Gramlich et al. (2009, loc. Cit.) ,

To test the effectiveness of AO treatment on adult animals, DOM was first induced by angiotensin II. Part of the animals received parallel intravenous treatment with the AO; The treatment with the AO with the sequence SEQ ID No. 3 is shown here by way of example. Cardiac function was checked regularly by means of echocardiography. The experimental setup is in the 4A illustrated.

shows echocardiographic data of this animal experiment. The treatment of animals with AD prevents the development of DOM in heterozygous titin mice, which is reflected in the normalization of the pumping function (fractional shortening) and the end-diastolic diameter (LVEDD).

In is shown the histological workup of the heart. While untreated animals develop pronounced myocardial fibrosis, as clearly visible in the Trichrom-Masson staining (left), this is barely visible in the animals treated with the AO (right).

This experiment shows that the AO therapy is possible and very effective on the adult mouse model and that the animals are not harmed by the treatment.

Example 7 - Human exon skipping

In order to be able to test the treatment with the AOs according to the invention in humans, patient-specific induced pluripotent stem cells, so-called iPS cells, were produced and these were transfected with the AOs with the sequences SEQ ID 8 to 13. 5 shows schematically the experimental setup.

A skin biopsy was taken of a patient carrying a mutation in exon 326 of titing. The affected patient has a severe DCM with severe heart failure. From the skin biopsy fibroblasts were isolated and cultured. The fibroblast culture was transfected with the so-called Yamanaka factors KLF4, SOX2, OCT4 and c-myc. This causes the reprogramming of the fibroblasts into pluripotent stem cells, the iPS cells. The iPS cells can now be differentiated into all subordinate cell types.

The iPS cells were differentiated into cardiomyocytes so that a patient-specific cardiomyocyte line is now available in culture carrying the titin mutation. The cells were transfected with the AOs with the sequences SEQ ID 8 to 13 and checked for successful skipping by RT-PCR.

This experiment shows that AO therapy is also possible on humans.

QUOTES INCLUDE IN THE DESCRIPTION

This list of the documents listed by the applicant has been generated automatically and is included solely for the better information of the reader. The list is not part of the German patent or utility model application. The DPMA assumes no liability for any errors or omissions.

Cited patent literature

• EP 1191097 [0006]
• EP 1619249 [0006]
• WO 2006/000057 [0006]
• EP 1857548 [0006]
• WO 2010/048586 [0006]
• US 2012/0041050 [0006]
• US 2012/0022134 [0006]

Cited non-patent literature

• Cirak et al. (2011), exon skipping and dystrophin restoration in patients with Duchenne muscular dystrophy after systemic phosphorodiamidate morpholino oligomer treatment: at open-label, phase 2, dose-escalation study, Lancet, Vol. 378, pages 595 to 605 [0006]
• Gerull et al. (2002), Mutations of TTN, encoding the giant muscle filament titin, cause familial dilated cause familial dilated cardiomyopathy, Nat. Genet. 30, pages 201 to 204 [0018]
• Gerull et al. (2002, loc. Cit.) [0019]
• Sazani et al. (2011), Chemical and mechanical toxicology evaluation of exon skipping phosphorodiamidate morpholino oligomers in mdx mice, Int. J. Toxicol. 30, pages 322 to 333 [0025]
• Kibbe et al. (2000), Handbook of Pharmaceutical Excipients, Third Edition, American Pharmaceutical Association and Pharmaceutical Press [0033]
• Gerull et al. (2002, supra). [0047]
• Gramlich et al. (2009), Stress-induced dilated cardiomyopathy in a knock-in mouse model mimicking human titin-based disease, Journal of Molecular and Cellular Cardiology 47, pp. 352-358 [0048]
• Gramlich et al. (2009, supra) [0061]

Claims (13)

An isolated antisense oligonucleotide (AO) having a sequence that is specifically hybridizable to a mRNA splicing sequence of a mutation-carrying exon of the pre-mRNA of the titin gene and that is capable of inducing skipping of that exon. Isolated AO according to claim 1, characterized in that the sequence is specifically hybridizable to the mRNA splicing sequence of the exon 326th An isolated AO according to claim 1 or 2, having a length of about 5 to 50, preferably from about 10 to about 40, more preferably from about 15 to about 30, and most preferably from about 19 to about 22 Having nucleotides. Isolated AO according to any one of claims 1 to 3, characterized in that the mRNA splicing sequence is an exonic splicing enhancer sequence. An isolated AO according to any one of claims 1 to 4 comprising morpholino oligonucleotides and / or 2'-O-methyl phosphorothioate oligonucleotides. An isolated AO according to any one of claims 1 to 5 having a nucleotide sequence specific to at least about 5, preferably at least about 10, more preferably at least about 15, most preferably at least about 19 continuous nucleotides of the sequence SEQ ID Nos. 1 and / or 2 can hybridize. An isolated AO according to any one of claims 1 to 6, characterized in that it has a nucleotide sequence which is selected from the group consisting of: SEQ ID Nos. 3 to 13. An isolated AO according to any one of claims 1 to 7 for the treatment of heart disease, preferably dilated cardiomyopathy. A pharmaceutical composition comprising the isolated AO of any one of claims 1 to 8 and a pharmaceutically acceptable carrier. Use of the isolated AO according to one of claims 1 to 8 for the specific inhibition of an mRNA splicing sequence, preferably an exonic splicing enhancer sequence, of a mutation-carrying exon of the pre-mRNA of the titin gene, preferably of the exon 326 of the titin Gene to induce skipping of the exon. Use of the isolated AO according to any one of claims 1 to 8 or the pharmaceutical composition according to claim 9 for the treatment of heart disease, preferably of dilated cardiomyopathy. A method of inducing a skipping of a mutation-bearing exon of the pre-mRNA of the titin gene, preferably of exon 326, which comprises administering the isolated AO of any one of claims 1 to 8 or the pharmaceutical composition of claim 9 into a biological cell or animal , A method of treating dilated cardiomyopathy comprising administering the isolated AO of any one of claims 1 to 8 or the pharmaceutical composition of claim 9 to a subject afflicted with a heart disease, preferably dilated cardiomyopathy.

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